Stem cells that transform to beating cardiomyocytes

ABSTRACT

Disclosed herein is a novel isolated population of stem cells, called spoc cells, that can be induced, either in vivo or in vitro, to differentiate into cardiomyocytes. Methods are disclosed herein to differentiate the spoc cells, and to utilize these spoc cells for screening agents that affect cardiomyocytes. Methods are also provided herein to utilize spoc cells in therapeutic applications for the treatment of myocardial defects, such as areas of ischemic or traumatic damage.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.10/863,004, filed Jun. 7, 2004, which is a continuation of U.S. patentapplication Ser. No. 10/003,400, filed Oct. 22, 2001, now abandoned.Both applications are incorporated herein in their entirety.

FIELD

This application relates to the field of stem cells, specifically tomethods of producing and differentiating muscle stem cells.

BACKGROUND

Many Americans die each year of congestive heart failure. Heart failuremay occur from a variety of causes, including cardiomyopathy, myocardialischemia, congenital heart disease, and valvular heart disease,resulting in cardiac cell death and myocardial dysfunction. Ascardiomyocytes are not replaced in adult myocardial tissue, physiologicdemands on the existing, healthy, cardiomyocytes leads to theirhypertrophy. Heart transplants have been the only recourse for patientsin end-stage heart disease, however the United Network of Organ Sharing(UNOS), has reported that although more than 40,000 patients werewaiting for heart transplants as of February 2000, only 2,345 peoplereceived a donated heart in 1998. Furthermore, heart transplants arecomplicated by the incompatibility between the transplanted donor tissueand the recipient's immune system, which requires life-longimmunosuppression. Yet another drawback of heart transplants is theirhigh cost.

An alternative approach to heart transplantation is to generatecardiomyocytes from stem cells in vitro that can be used in thetreatment of heart failure, and other cardiac diseases characterized bymyocardial cell death or dysfunction. This approach is based on theability of stem cells to both self-renew and differentiate into one ormore mature cell types, including cardiomyocytes. Stem cells may beobtained from an individual suffering from heart disease and then usedto generate cardiomyocytes in vitro in order to repair the damagedmyocardium. This approach avoids problems inherent with hearttransplantation, such as lack of a suitable heart for transplant orimmune rejection of a transplanted heart.

Embryonic stem (ES) cells, derived from the inner cell mass of theblastocyst, are the most primitive stem cell, as disclosed in WO01/11011 A2. These cells have unlimited self-remewal capability, andbecause they can differentiate into several cell lineages and repopulatetissues upon transplantation, they have multipotent differentiativepotential. However, protocols are not available for differentiatingembryonic stem cells into beating cardiomyocytes.

Lineage specific stem cells, identified in most organ tissues, have lessself-renewal capability than ES cells and their differentiative abilityis limited to tissues of that lineage. Of the lineage specific stemcells, the hematopoietic stem cell (HSC), derived from bone marrow,blood, cord blood, fetal liver and yolk sack, is the best characterized.These cells are defined by the expression of cell surface markers, suchas c-kit (c-kit+), and can terminally differentiate into all thehematopoietic cell types. HSC have been shown to contribute to theformation of functional cardiac tissue in vivo (Jackson et al , J. Clin.Invest., 107:1395-1402, 2001). Mesenchymal stem cells (MSC) arepluripotent progenitor cells derived from tissues of mesodermal origin(U.S. Pat. No. 5,486,359). These cells are most often obtained from bonemarrow, although they can be obtained from other sources, such as bloodor dermis. These cells have been shown to differentiate to form muscle,bone, cartilage, fat, marrow stroma and tendon, but have not been shownto differentiate into cardiomyocytes. In addition, progenitor cells havebeen identified in skeletal muscle, termed satellite cells (Cornelisonand Wold, Dev. Biol., 191:270-283, 1997). These cells are characterizedby the expression of the cell surface marker c-met (c-met+) in both itsquiescent and activated states. When activated these cells re-enter thecell cycle, express myogenic regulatory factors, and differentiate intomyoblasts.

However, despite the existence of a variety of stem cells, there iscurrently no pure population of stem cells that can be induced underdefined conditions to differentiate into spontaneously beatingcardiomyocytes in vitro. Thus, there remains a need in the art forisolated populations of stem cells which can be induced to differentiateinto cardiomyocytes.

SUMMARY

The methods and cells described herein are based on the ability ofcertain stem cells to be differentiated in vitro to form a fullyfunctional cell of more than one given cell type.

Disclosed herein is a novel isolated population of stem cells, calledspoc cells, that can be induced, either in vivo or in vitro, todifferentiate into cardiomyocytes. Methods are disclosed herein todifferentiate the spoc cells, and to utilize these spoc cells forscreening agents that affect cardiomyocytes. Methods are also providedherein to utilize spoc cells in therapeutic applications.

The foregoing and other objects, features, and advantages will becomemore apparent from the following detailed description of a severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of digital images of transmission electronmicrographs demonstrating the progression of differentiation of CS(cardiac precursor from spoc cells) cells over time when cultured indifferentiation medium. FIG. 1A is a digital image of CS cell at day 3with disordered myosin filaments. FIG. 1B is a digital image showingthat at day 7 myosin filaments of characteristic 1.6 μm-length (top box)radiate outward and the cells contain dense bodies (lower box). FIG. 1Cand FIG. 1F are digital images of a cell at day 14, showing a single,central nucleus shows a stretching out of the dense bodies into anorganizing sarcomere. FIG. 1D shows that day 3 CS cells are round cellswith copious mitochondria (box and detail). FIG. 1E shows elongated day7 cells contain dense bodies (arrowhead). FIG. 1G shows that by day 56,a well-defined sarcomere (FIG. 1G) is present, with identifiable A- andI-bands and Z-lines.

FIG. 2 demonstrates the existence of calcium transients, incardiomyocytes differentiated from CS cells. FIG. 2A shows a graphicalrepresentation of the calcium transient in a beating CS cell-derivedcardiomyocyte. Peak intensity and baseline are shown in FIG. 2B and FIG.2C, respectively.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

In order to facilitate review of the various embodiments disclosedherein, the following list of abbreviations and explanation of terms isprovided:

I. Abbreviations and Terms

-   A. Abbreviations-   CS: Cardiac precursors from spoc cells-   DNA: Deoxyribonucleic acid-   EGF: Epidermal growth factor-   EGFP: Enhanced green fluorescent protein-   ES: Embryonic stem-   FACS: Fluorescence activated cell sort-   FBS: Fetal bovine serum-   FGF: Fibroblast growth factor-   HSC: Hematopoietic stem cell-   MRNA: Messenger ribonucleic acid-   PBS: Phosphate buffered saline-   RNase: Ribonuclease-   RT-PCR: Reverse transcriptase-polymerase chain reaction-   SPOC: Skeletal-based precursors of cardiomyocytes

B. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VI, published by Oxford UniversityPress, 1997 (ISBN 0-19-857778-8); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8)

Adult: A fully developed and physically mature subject, having attainedfull size and strength.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds.

Cardiac: Pertaining to the heart.

Cardiac dysfunction: Any impairment in the heart's pumping function.This includes, for example, impairments in contractility, impairments inability to relax (sometimes referred to as diastolic dysfunction),abnormal or improper functioning of the heart's valves, diseases of theheart muscle (sometimes referred to as cardiomyopathy), diseases such asangina and myocardial ischemia and infarction characterized byinadequate blood supply to the heart muscle, infiltrative diseases suchas amyloidosis and hemochromatosis, global or regional hypertrophy (suchas may occur in some kinds of cardiomyopathy or systemic hypertension),and abnormal communications between chambers of the heart (for example,atrial septal defect). For further discussion, see Braunwald, HeartDisease: a Textbook of Cardiovascular Medicine, 5th edition 1997, WBSaunders Company, Philadelphia Pa. (hereinafter Braunwald).

Cardiac muscle: The heart is made of specialized muscle tissue with somesimilarities to both smooth and skeletal muscle. It is involuntary andmononucleate as is smooth muscle. Cardiac muscle is striated likeskeletal muscle, which means that it has microscopically visiblemyofilaments arranged in parallel with the sarcomere. These filamentsslide along each other during the process of contraction in the samemanner as occurs in skeletal muscle. However, cardiac muscle containsmore mitochondria so the striations are not as organized as they are inskeletal muscle. Cardiac muscle also differs from skeletal muscle inthat the fibers in cardiac muscle branch and usually have a singlecentrally located nucleus. Another difference in cardiac muscle is thepresence of intercalated discs which serve as specialized connectionsbetween cardiac muscle cells. These tight connections allow for almostcompletely free movement of ions so that action potentials can freelypass from one cell to another. This arrangement makes cardiac muscletissue a functional syncytium. When one cell is excited the resultantaction potential is spread to all of them. This is an important featurein that it allows the atrial or ventricular muscle to contract as a unitto forcefully pump blood. Cardiac muscle can generate its own excititoryimpulses from the sino-atrial node, which acts like a biologicalpacemaker. In this manner, the contracting signal for cardiac musclesoriginates in the heart itself However, the autonomic nervous system(for example through the vagus nerve) can exert control over how fastthe signals form and propagate through the heart, which regulates therate of myocardial contraction. A “cardiomyocyte” is a cell of thecardiac muscle.

Cardiac precursors from spoc cells (CS cells): When spoc cells areisolated from skeletal muscle and are cultured under growth conditionsdesigned to promote their growth, spoc cells undergo several rounds ofdivision. During this proliferative phase they become clusters offloating round cells with an increased diameter as compared to spoccells. These round cells, with an increased diameter, are referred to asCS cells. In one embodiment, a diameter of a CS cell is from about 10 toabout 14 μm. When placed in growth promoting conditions in vitro (suchas the examples described below) CS cells differentiate intospontaneously beating cardiomyocytes.

Cardiomyopathy: Any disease or dysfunction of the myocardium (heartmuscle) in which the heart is abnormally enlarged, thickened and/orstiffened. As a result, the heart muscle's ability to pump blood isusually weakened. The disease or disorder can be, for example,inflammatory, metabolic, toxic, infiltrative, fibroplastic,hematological, genetic, or unknown in origin. There are two generaltypes of cardiomyopathies: ischemic (resulting from a lack of oxygen)and nonischemic. Ischemic cardiomyopathy is a chronic disorder caused bycoronary artery disease—a disease in which there is atheroscleroticnarrowing or occlusion of the coronary arteries on the surface of theheart. Coronary artery disease often leads to episodes of cardiacischemia, in which the heart muscle is not supplied with enoughoxygen-rich blood. Eventually, the heart muscle enlarges from theadditional work it must do in the absence of sufficient oxygen-richblood.

Nonischemic cardiomyopathy is generally classified into three groupsbased primarily on clinical and pathological characteristics:

-   -   (1) dilated cardiomyopathy, a syndrome characterized by cardiac        enlargement and impaired systolic function of one or both        ventricles;    -   (2) hypertrophic cardiomyopathy, herein defined as (a) global or        regional increase in thickness of either ventricular wall or the        interventricular septum, or (b) an increased susceptibility to        global or regional increase in thickness of either ventricular        wall or the interventricular septum, such as can occur in        genetic diseases, hypertension, or heart valve dysfunction; or    -   (3) restrictive and infiltrative cardiomyopathies, a group of        diseases in which the predominant clinical feature is usually        impaired ability of the heart to relax (diastolic dysfunction),        and is often characterized by infiltration of the heart muscle        with foreign substances such as amyloid fibers, iron, or        glycolipids.        See Wynne and Braunwald, The Cardiomyopathies and        Myocarditities, Chapter 41, supra.

Cell surface marker: A protein, glycoprotein, or other moleculeexpressed on the surface of a cell, which serves to help identify thecell. A cell surface marker can generally be detected by conventionalmethods. Specific, non-limiting examples of methods for detection of acell surface marker are immunohistochemistry, fluorescence activatedcell sorting (FACS), or an enzymatic analysis.

Congenital heart disease: A heart-related problem that is present sincebirth and often as the heart is forming even before birth. Congenitalheart disease may affect the heart, the heart's valves, the veinsleading to, or the arteries leading away, from the heart, or theconnections between these parts of the body.

Differentiation: The process whereby relatively unspecialized cells(e.g., stem cells) acquire specialized structural and/or functionalfeatures characteristic of mature cells. Similarly, “differentiate”refers to this process. Typically, during differentiation, cellularstructure alters and tissue-specific proteins appear. The term“differentiated muscle cell” refers to cells expressing a proteincharacteristic of the specific muscle cell type. A differentiated musclecell includes a skeletal muscle cell, a smooth muscle cell, and acardiac muscle cell.

Differentiation Medium A synthetic set of culture conditions with thenutrients necessary to support the growth or survival of cultured cells,and which allows the differentiation of stem cells into differentiatedcells.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide. Theterm codon is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Epidermal growth factor (EGF): In particular examples, EGF is a globularprotein of 6.4 kDa consisting of 53 amino acids. It contains threeintramolecular disulfide bonds essential for biological activity. EGFproteins are evolutionarily closely conserved. Human EGF and murine EGFhave 37 amino acids in common. Approximately 70 percent homology isfound between human EGF and EGF isolated from other species. MammalianEGF includes, but is not limited to, murine, avian, canine, bovine,porcine, equine, and human EGF. The amino acid sequences and methods formaking these EGF polypeptides are well known in the art.

The gene encoding the EGF precursor has a length of approximately 110kb, and contains 24 exons. Fifteen of these exons encode protein domainsthat are homologous to domains found in other proteins. The human EGFgene maps to chromosome 4q25-q27.

EGF is a strong mitogen for many cells of ectodermal, mesodermal, andendodermal origin. EGF controls and stimulates the proliferation ofepidermal and epithelial cells, including fibroblasts, kidney epithelialcells, human glial cells, ovary granulosa cells, and thyroid cells invitro. EGF also stimulates the proliferation of embryonic cells.However, the proliferation of some cell lines has been shown to beinhibited by EGF.

EGF is also known to act as a differentiation factor for some celltypes. It strongly influences the synthesis and turn-over of proteins ofthe extra-cellular matrix including fibronectin, collagen, laminin, andglycosaminoglycans, and has been shown to be a strong chemoattractantfor fibroblasts and epithelial cells.

EGF can be assayed in a cell-based assay wherein the proliferation of acell population is assessed. EGF can also be assayed by an immunoassay,such as an ELISA assay.

Fragments of EGF, smaller than the full-length sequence can also beemployed in methods disclosed herein. Suitable biologically activevariants can also be utilized. One specific, non-limiting example of anEGF variant of use is an EGF sequence having one or more amino acidsubstitutions, insertions, or deletions, wherein a biological functionof EGF is retained. Another specific, non-limiting example of an EGFvariant is EGF as wherein glycosylation or phosphorylation is altered,or a foreign moiety is added, so long as a biological function of EGF isretained. Methods for making EGF fragments, analogues, and derivativesare available in the art. Examples of EGF variants are known in the art,for example U.S. Pat. No. 5,218,093 and WO 92/16626A1. Examples of EGFfrom many different species are disclosed in WO 92/16626A1, as areexamples of variants, and strategies for producing them.

As used herein, “EGF” refers to naturally occurring EGF, and variantsand fragments that perform the same function of EGF in the culture mediadisclosed herein.

Embryonic stem (ES) cells are totipotent cells isolated from the innercell mass of the developing blastocyst and can generate all of the cellspresent in the body (bone, muscle, brain cells, etc.). “ES cells” can bederived from any organism, for example from mammals such as humans.

Fibroblast growth factor (FGF): Any suitable fibroblast growth factor,derived from any animal, and functional variants and fragments thereof.A variety of FGFs are known and include, but are not limited to, FGF-1(acidic fibroblast growth factor), FGF-2 (basic fibroblast growthfactor, bFGF), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6, FGF-7,FGF-8, and FGF-9. FGF refers to a fibroblast growth factor protein suchas FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, or FGF-9, or a biologically activefragment or mutant thereof. The FGF can be from any animal species. Inone embodiment the FGF is mammalian FGF including but not limited to,rodent, avian, canine, bovine, porcine, equine, and human. The aminoacid sequences and method for making many of the FGFs are well known inthe art.

Fragments of FGF that are smaller than those described can also beemployed.

Suitable biologically active variants can be FGF analogues orderivatives. An analogue of FGF is either FGF or an FGF fragment thatincludes a native FGF sequence and structure having one or more aminoacid substitutions, insertions, or deletions. Analogs having one or morepeptoid sequences (peptide mimic sequences) are also included (see e.g.International Publication No. WO 91/04282). By “derivative” is intendedany suitable modification of FGF, FGF fragments, or their respectiveanalogues, such as glycosylation, phosphorylation, or other addition offoreign moieties, so long as the FGF activity is retained. Methods formaking FGF fragments, analogues, and derivatives are available in theart.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. In general, growth factors stimulate cell proliferationor maturation when they bind to their receptor. In one embodiment,growth factors are a complex family of polypeptide hormones orbiological factors that control growth, division, and maturation ofmuscle cells. In another embodiment a growth factor can be used topromote the proliferation of muscle stem cells and maintain the stemcells in an undifferentiated state. A growth factor can be a naturallyoccurring factor or a factor synthesized using molecular biologytechniques. Examples of growth factors include platelet-derived growthfactor, fibroblast growth factor, epidermal growth factor, insulin,somatomedin, stem cell factor, vascular endothelial growth factor,granulocyte colony stimulating factor, and transforming growthfactor-beta, amongst others. A muscle cell growth factor is a growthfactor that effects the development (maturation), differentiation,division, or proliferation of muscle cells.

Growth medium: A synthetic set of culture conditions with the nutrientsnecessary to support the growth or survival of microorganisms or culturecells.

Heart: The muscular organ of an animal that circulates blood. The wallsof the heart are comprised of working muscle, or myocardium, andconnective tissue. Myocardium is comprised of myocardial cells, whichare also referred to herein as cardiac cells, cardiac myocytes,cardiomyocytes and/or cardiac fibers. Cardiomyocytes may be cells of theatrium or cells of the ventricle.

Heart failure: The inability of the heart to supply sufficientoxygenated blood to meet the metabolic needs of the tissues and cells ina subject. This can be accompanied by circulatory congestion, such ascongestion in the pulmonary or systemic veins. As used herein, the termheart failure encompasses heart failure from any cause, and is intendedherein to encompass terms such as “congestive heart failure,” “forwardheart failure,” “backward heart failure,” “high output heart failure,”“low output heart failure,” and the like. See Chapters 13-17 inBraunwald for a detailed discussion. Conditions that could lead to heartfailure include, but are not limited to, coronary artery disease,cardiomyopathy, or congenital heart disease.

Heterologous: A heterologous sequence is a sequence that is not normally(i.e. in the wild-type sequence) found adjacent to a second sequence. Inone embodiment, the sequence is from a different genetic source, such asa virus or organism, than the second sequence.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Muscle cell: Includes skeletal, cardiac or smooth muscle tissue cells.This term is synonymous with myocyte, and encompasses those cells whichdifferentiate to form more specialized muscle cells (e.g. myoblasts).“Cardiomyocyte” refers to a cardiac muscle cell.

Myocardial injury: Damage to the muscle or the “myocardium” in the wallof the heart as a result of disease or trauma. Myocardial injury can beattributed to many things such as, but not limited to, cardiomyopathy,myocardial infarction, or congenital heart disease.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Pharmaceutically acceptable carriers: Remington's PharmaceuticalSciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15thEdition (1975), describes compositions and formulations suitable forpharmaceutical delivery of stem cells herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent: Refers to a chemical compound or compositioncapable of inducing a desired therapeutic or prophylactic effect whenproperly administered to a subject or a cell. “Incubating” includesexposing a target to an agent for a sufficient period of time for theagent to interact with a cell. “Contacting” includes incubating an agentin solid or in liquid form with a cell.

Polypeptide refers to a polymer in which the monomers are amino acidresidues which are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred. The terms“polypeptide” or “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those which are recombinantlyor synthetically produced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide. Biologically functionalfragments, for example, can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. An “epitope” is aregion of a polypeptide capable of binding an immunoglobulin generatedin response to contact with an antigen. Thus, smaller peptidescontaining the biological activity of insulin, or conservative variantsof the insulin, are thus included as being of use.

The term “soluble” refers to a form of a polypeptide that is notinserted into a cell membrane.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Examples ofconservative substitutions are shown below. Original ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys SerGln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; GluMet Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe ValIle; Leu

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, are usually minimized in order to preservethe functional and immunologic identity of the encoded protein. Theimmunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody; a variant that is recognized bysuch an antibody is immunologically conserved. Any cDNA sequence variantwill preferably introduce no more than twenty, and preferably fewer thanten amino acid substitutions into the encoded polypeptide. Variant aminoacid sequences may, for example, be 80, 90 or even 95% or 98% identicalto the native amino acid sequence. Programs and algorithms fordetermining percentage identity can be found at the NCBI website.

Precursor Cell: A cell that can generate a fully differentiatedfunctional cell of at least one given cell type. Generally, precursorcells can divide. After division, a precursor cell can remain aprecursor cell, or may proceed to terminal differentiation. A “muscleprecursor cell” is a precursor cell that can generate a fullydifferentiated functional muscle cell, such as a cardiomyocyte or askeletal muscle cell. One specific, non-limiting example of a muscleprecursor cell is a “cardiac precursor cell,” which is a cell that givesrise to cardiac muscle cells.

Progenitor Cell: A cell that gives rise to progeny in a defined celllineage. A “muscle progenitor cell” is a cell that gives rise to cellsof the muscle lineage. One specific, non-limiting, example of a skeletalmuscle progenitor cell is a “satellite cell,” which gives rise toimmature and mature skeletal muscle cells.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Similarly, a recombinant protein is one encoded for by a recombinantnucleic acid molecule.

Skeletal muscle: Skeletal muscle makes up most of the body's muscle anddoes not contract without nervous stimulation. It is under voluntarycontrol and lacks anatomic cellular connections between fibers. Thefibers (cells) are multinucleate and appear striated due to thearrangement of actin and myosin protein filaments. Each fiber is asingle cell, long, cylindric and surrounded by a cell membrane. Themuscle fibers contain many myofibrils that are made of myofilaments.These myofilaments are made up of contractile proteins. The key proteinsin muscle contraction are myosin, actin, tropomyosin and troponin.

Skeletal-based precursor of cardiomyocytes (Spoc) cells: Stem cellsderived from skeletal muscle, which do not express the cell surfacemarkers c-met, or c-kit, that can be differentiated into cardiomyocytes.In one embodiment spoc cells are muscle derived precursor cells that areabout 4 μm in diameter when cultured in vitro. These cells remain insuspension and proliferate when cultured in the presence of a growthfactor. Specific, non-limiting examples of growth factors of use inpropagating spoc cell are FGF, EGF, or a combination thereof.

In one embodiment, spoc cells differentiate into spontaneously beatingcardiomyocytes in vitro. During a proliferative phase (e.g. about 7 daysafter being maintained in vitro in the presence of a growth factor),spoc cells cluster and increase in size to about 10-14 μm in diameter.The cells in these clusters, referred to as CS cells, have the abilityto differentiate into mature cardiac muscle cells when cultured in theabsence of growth factors. Methods for isolating and differentiatingspoc cells are disclosed herein.

Spontaneous: arising from an internal cause, resulting from internal ornatural processes, with no apparent external influence. A “spontaneouslybeating cardiomyocyte” is a cell that begins to beat as a result ofinternal signals.

Stem cell refers to a cell that can generate a fully differentiatedfunctional cell of more than one given cell type. The role of stem cellsin vivo is to replace cells that are destroyed during the normal life ofan animal. Generally, stem cells can divide without limit. Afterdivision, the stem cell may remain as a stem cell, become a precursorcell, or proceed to terminal differentiation. Although appearingmorphologically unspecialized, the stem cell may be considereddifferentiated where the possibilities for further differentiation arelimited. A “muscle stem cell” is a stem cell derived from muscle or thatgives rise to muscle cells after differentiation. One specific,non-limiting example of a muscle stem cell is a cell that gives rise tocardiac muscle cells.

Subject refers to any mammal, such as humans, non-human primates, pigs,sheep, cows, rodents and the like which is to be the recipient of theparticular treatment. In one embodiment, a subject is a human subject ora murine subject.

Suspension: a dispersion of solid particles, such as a cell, throughoutthe body of a liquid, such as a culture medium or an isotonic(physiologically compatible) buffer.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents.

Therapeutically effective amount is the amount of agent that issufficient to prevent, treat, reduce and/or ameliorate the symptomsand/or underlying causes of any of a disorder or disease. In oneembodiment, a “therapeutically effective amount” is sufficient to reduceor eliminate a symptom of a cardiac disease. In another embodiment, atherapeutically effective amount is an amount sufficient to overcome thedisease itself.

A therapeutically effective amount of a cell can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of the cells will be dependenton the subject being treated, the severity and type of the condition,and the manner of administration of the compound. “Administering” can beaccomplished by introducing the therapeutically effective amount locallyor systemically into the subject. Systemic introduction can beaccomplished by using an intravenous, intramuscular, transcutaneous orsubcutaneous means. Such means could include introducing thetherapeutically effective amount via injection, or via catheter.

The general term “administering a therapeutically effective amount tothe subject” is understood to include all animals (e.g. humans, apes,dogs, cats, horses, and cows) that have or may develop some form ofcardiac dysfunction.

Transfected: A transfected cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transductionwith viral vectors, transformation with plasmid vectors, andintroduction of DNA by electroporation, lipofection, and particle gunacceleration.

Transplantation: The transfer of a tissue or an organ, or a portionthereof, from one body or part of the body to another body or part ofthe body.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used, suitable methods and materials aredescribed below. In case of conflict, the present specification,including the explanation of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Spoc Cells

Stem cells derived from skeletal muscle (spoc cells) are disclosedherein. Spoc cells do not express the cell surface markers c-met, orc-kit, and are thus termed a c-met⁻/c-kit⁻ cell. Spoc cells can beisolated from any age mammal, either human or non-human. Thus spoc cellscan be obtained from a fetus, a child or an adult of any mammalianspecies. In one embodiment, a spoc cell is human or murine c-met⁻/c-kit⁻cell that can be differentiated into a cardiomyocyte in vitro. In oneembodiment, the spoc cell is between about 3μm and 10 μm in diameter, orare about 4 μm in diameter.

Culture conditions for spoc cells have been identified and are disclosedherein. In one embodiment, spoc cells do not adhere to the culture dishbut remain in suspension when cultured in the presence of at least onegrowth factor. In one specific, non-limiting example, the growth factoris EGF. In another specific, non-limiting example, the growth factor isFGF.

Culture conditions are also disclosed herein (see below) fordifferentiating spoc cells. The differentiation of spoc cells intocardiomyocytes can be assessed by observing morphological changes. Insome examples, differentiated spoc cells are spontaneously beatingcardiomyocytes. In several embodiments, organized gap junctions andsarcomeres with clear Z-lines and A- and I-bands, are observed in thedifferentiated spoc cells. In addition, certain examples of thedifferentiated spoc cells may be mono- or multi-nucleate. In oneembodiment the cells are bi-nucleate.

The isolated spoc cell can be transduced using standard procedures knownin molecular biology in order to introduce a nucleic acid molecule ofinterest into the cell. In one embodiment, the nucleic acid moleculeencodes a polypeptide. The polypeptide encoded by the nucleic acidmolecule can be from the same species as the cells (homologous), or canbe from a different species (heterologous). For example, a nucleic acidmolecule can be utilized that supplements or replaces deficientproduction of a peptide by the tissue of the host wherein suchdeficiency is a cause of the symptoms of a particular disorder. In thiscase, the cells act as a source of the peptide. In one specific,non-limiting example the polypeptide is the cardiac specifictranscription factor GATA-4.

In one embodiment, the nucleic acid molecule of interest encodes apolypeptide involved in growth regulation or neoplastic transformationof cardiac cells. Specific, non-limiting examples of nucleic acidssequences of interest are SV40 Tag, p53, myc, src, and bc1-2. In anotherembodiment, the nucleic acid sequence of interest encodes an enzyme.Specific, non-limiting examples of enzymes are proteins involved in theconversion of a pro-drug to a drug, or growth factors that promote theexpansion, differentiation, or survival of cardiac progenitor cells,such as EGF, FGF, or atrial natriuretic factor. In yet anotherembodiment, the nucleic acid sequence of interest encodes atranscriptional regulator.

In one embodiment, the nucleic acid sequence of interest is operablylinked to a regulatory element, such as a transcriptional and/ortranslational regulatory element. Regulatory elements include elementssuch as a promoter, an initiation codon, a stop codon, mRNA stabilityregulatory elements, and a polyadenylation signal. A promoter can be aconstitutive promoter or an inducible promoter. Specific non-limitingexamples of promoters include the CMV promoter, an atrial natriureticfactor promoter, and promoters including TET-responsive element forinducible expression of transgene. In another embodiment, the nucleicacid sequence of interest is inserted into a vector, such as anexpression vector. Procedures for preparing expression vectors are knownto those of skill in the art and can be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989). Expression of the nucleic acidof interest occurs when the expression vector is introduced into anappropriate host cell.

In yet another specific, non-limiting example, a nucleic acid sequencecan be introduced to decrease rejection. For example, the immunogenicityof a cell may be suppressed by deleting genes that produce proteins thatare recognized as “foreign” by the host (a knock-out), or by introducinggenes which produce proteins, such as proteins that are native to thehost and recognized as “self” proteins by the host immune system.

Thus in one embodiment, a spoc cell may be transfected with a nucleicacid molecule designed to functionally delete or “knock-out” a gene ofinterest. In this method, the nucleic acid molecule of interest is anucleic acid molecule that undergoes homologous recombination and isinserted into the genome of the spoc cell. Methods for producing“knock-outs” in ES cells are known to one of skill in the art (e.g. seeU.S. Pat. No. 5,939,598).

According to this example, cells are cultured in vitro as describedherein and an exogenous nucleic acid is introduced into the cells by anymethod known to one of skill in the art, for example, by transfection orelectroporation. The transfected cultured cells can then be studied invitro or can be administered to a subject (see below). Methods for theintroduction of nucleic acid sequences into stem cells are known in theart (e.g., see U.S. Pat. No. 6,110,743).

Methods of Isolating and Expanding Muscle Stem Cells

A method of isolating a c-met⁻/c-kit⁻ cardiomyocyte precursor cell ofmuscular origin (spoc cell) is described herein. In this method, spoccells are separated by size from a suspension of muscle cells and thecells are cultured on a solid substrate. The cells that remain insuspension in the culture medium are isolated.

The method of isolation of the spoc cells includes obtaining the cellsfrom the muscle of a subject. Muscle tissue can be prepared for thepurpose of isolating or obtaining individual spoc cells by using methodswell known to one of skill in the art. Examples of methods of tissuepreparation include enzymatic digestion with enzymes such ascollagenase, mechanical disruption using instruments such as hand-heldor motor-driven homogenizers, or by chemical disruption using, forexample, chelators of calcium and magnesium.

The preparation of muscle cells can be sorted by any method thatseparates cells on the basis of cell size. In one embodiment, the spoccells are isolated by passing digested skeletal muscle through a seriesof filters of varying pore size. The cells are passed through twofilters, where a first filter has a pore size of about 50-200 μm, about60-150 μm, about 80-100 μm, or about 100 μm and a second filter has apore size of about 10-50 μm, 20-40 μm, or about 40 μm. In one embodimentthe isolated cells are less than 40 μm in diameter. In otherembodiments, isolated cells are between about 3 μm and 10 μm indiameter. In another embodiment the isolated cells are about 4 μm indiameter.

The cells can be also sorted by size by passing them throughsize-exclusion columns. In one such embodiment, the cells are elutedalong a size gradient such that the largest cells are eluted first andthe smallest cells are eluted last. The cells can also be sorted by sizeusing FACS. Cells of about 3 μm to 10 μm in diameter, or of about 4 μmin diameter, are isolated.

Once the muscle cells are sorted by size the cells are further selectedand then expanded in culture medium. In one embodiment the cells arecultured on a solid substrate that permits the adhesion of asubpopulation of cells in the presence of a culture medium. In oneembodiment, the solid substrate is a container, such as a tissue culturedish. In another embodiment, the solid substrate is in the form of beadsdesigned for tissue culture. The medium can be a growth medium, or anybuffer that maintains the viability of the cells. A variety of culturemedia are known and are suitable for use. Generally, the growth mediumincludes a minimal essential medium. In one embodiment, the medium isDMEM and/or F12, or a combination of DMEM and F12 (at a ratio betweenabout 1:1 to about 10:1).

The growth medium may be supplemented with serum. Specific, non-limitingexamples of serum are horse, calf or fetal bovine serum. The medium canhave between about 3% by volume to about 10% by volume serum, or about5% by volume serum.

In one embodiment, the medium contains one or more additional additivessuch as nutrients. Specific, non-limiting examples of these nutrientsare shown in the table below: Additive Exemplary Concentration serumAbout 3% to about 10% insulin About 5 μg/ml to about 10 μg/mltransferrin About 5 μg/ml to about 10 μg/ml selenium About 6 ng/mlethanolamine About 2 μg/ml EGF About 5 ng/ml to about 10 ng/ml FGF About5 ng/ml to about 10 ng/ml gentamycin About 25 μg/ml to about 50 μg/mlfungizone About 0.2 μg/ml to about 2.5 μg/ml

The muscle stem cell growth media can also be supplemented with growthfactors. In one embodiment, the growth medium includes basic fibroblastgrowth factor (bFGF). In one specific example, the growth mediumincludes between about 2 ng/ml to about 100 ng/ml of bFGF, such as forexample between about 5 ng/ml to about 50 ng/ml, between about 8 ng/mlto about 20 ng/ml, or between about 5 to about 10 ng/ml bFGF. In yetanother example, the medium includes about 10 ng/ml bFGF. In anotherembodiment, the growth medium includes epidermal growth factor (EGF). Inone specific example, the growth medium includes between about 2 ng/mlto about 100 ng/ml of EGF, such as for example between about 5 ng/ml toabout 50 ng/ml, between about 8 ng/ml to about 20 ng/ml, or betweenabout 5 ng/ml to about 10 ng/ml EGF. In yet another example, the mediumincludes about 10 ng/ml EGF. Thus in one embodiment, the growth mediumis 1:1 DMEM/F12 and includes 5% fetal bovine serum, 10 ng/ml FGF, 10ng/ml EGF, 5 μg/ml insulin, 5 μg/ml transferrin, 6 ng/ml selenium, 2μg/ml ethanolamine.

In one specific, non-limiting example the cells are cultured in thegrowth medium for about 4 days to about 8 days. In another specific,non-limiting example, the cells are cultured in the growth medium forabout 6 days to about 7 days.

During the period that the cells are cultured in the presence of growthfactors, the cells cluster and increase in size. Within the clusters thecells are between about 5-20 μm in diameter, or between about 10-14 μmin diameter.

A method is also provided for isolating spoc cells wherein the spoccells are identified using specific binding agents, such as antibodies,for example monoclonal antibodies that recognize cell surface markers.This particular method of isolation of the spoc cells includes obtainingthe cells from the muscle of a subject, as described above. In oneembodiment, the cells are selected by size (see above) and then thec-met⁻/c-kit⁻ spoc cells are identified using the specific bindingagents, such as antibodies that recognize the c-met and c-kit cellsurface markers.

In one embodiment the c-met and c-kit antibodies are immobilized. Aparticular embodiment uses magnetic cell sorting. This method involves acombination of monoclonal antibodies which are covalently bound to thesurface of magnetic beads and which are directed to cell surface markerswhich are absent from the cells being selected. For example, to isolatethe c-met⁻/c-kit⁻ spoc cells, monoclonal antibodies to c-met and c-kitbound to magnetic beads are used. All cells expressing either c-met, orc-kit, or both c-met and c-kit, will be bound by the antibodies andretained by the beads. Since the cells bound to the magnetic beads areimmobilized by the magnet, the c-met⁻/c-kit⁻ cells that remain insuspension can be isolated from the other cells.

In another embodiment, purified populations of c-met⁻/c-kit⁻ spoc cellsare isolated via FACS. Fluorescent-tagged antibodies against c-met andc-kit identify c-met+, c-kit+ and c-met+/c-kit+ double-positivepopulations of cells, allowing for the identification and isolation ofthe double-negative c-met⁻/c-kit⁻ population.

In other embodiments a single antibody, or a combination of antibodies,can be covalently bound to inert beads, such as sepharose beads. Thebeads can be packed in a column or maintained as a slurry. The cellsexpressing one or more of the cell surface markers are recognized by oneor more of the antibodies, thus becoming bound to the beads, therebyidentifying a subpopulation of unbound cells that does not express thecombination of cell surface markers.

In another embodiment the antibodies are not immobilized. In aparticular embodiment the addition of the antibodies to a mixture ofcells causes the aggregation of cells expressing the cell surfacemarkers recognized by the antibodies. The cells not expressing the cellsurface markers are excluded from the aggregates and can be isolated.

Spoc cells isolated by these or other methods can be maintained inculture. The spoc cells can further be differentiated intocardiomyocytes.

Methods of Differentiating Muscle Stem Cells

A method is disclosed herein for differentiating a spoc cell into acardiomyocyte. In a particular example, the cardiomyocyte is aspontaneously beating cardiomyocyte.

In one embodiment, differentiation into cardiomyocytes is induced byculturing cells in medium similar to the growth medium, but which doesnot include at least one growth factor. Thus, a specific, non-limitingexample of a differentiation medium is a growth medium that lacks atleast one growth factor. Growth factors removed from the medium include,but are not limited to, bFGF or EGF, or a combination of bFGF and EGF.

Removal of at least one growth factor causes the cells to adhere to thetissue culture dish and acquire characteristics of a differentiatedcardiomyocyte. Differentiation refers to the process whereby relativelyunspecialized cells, such as the c-met⁻/c-kit⁻ muscle-derived stem cellsacquire specialized structural and/or functional features characteristicof mature cells, such as cardiomyocytes.

Differentiation of c-met⁻/c-kit⁻ muscle stem cells into cardiomyocytes,such as spontaneously beating cardiomyocytes, can be measured by anymethod known to one of skill in the art. Specific, non-limiting examplesare immunohistochemical analysis to detect expression of cardiacpolypeptides (e.g. troponin-T, L-type calcium channel, orcardiac-specific transcription factors GATA-4, or Nkx2.5), or assayssuch as ELISA assay and Western blot analysis. Differentiation of cellscan also be measured by assaying the level of mRNA coding for cardiacpolypeptides using techniques such as Northern blot, RNase protectionand RT-PCR. In another embodiment, the number of spontaneously beatingcells is assessed.

Calcium transients, or the flux in intracellular calcium concentrations,can be used as a measure of cardiomyocyte differentiation. In oneembodiment calcium imaging is used to measure calcium transients. Forexample, ratiometric dyes, such as fura-2, fluo-3, or fluo-4 are used tomeasure intracelluar calcium concentration. The relative calcium levelsin a population of cells treated with a ratiometric dye can bevisualized using a fluorescent microscope or a confocal microscope. Inother embodiments, the membrane potential across the cell membrane ismonitored to assess calcium transients. For example, a voltage clamp isused. In this method, an intracellular microelectrode is inserted intothe cardiomyocyte.

In one embodiment, calcium transients can be seen before observablecontractions of the cardiomyocytes. In other embodiments calciumtransients are seen either during, or after, observable contractions ofcardiomyocytes. In another embodiment the cells are cultured in thepresence of conditions wherein the cells do not beat, such as in thepresence of a calcium chelator (e.g. EDTA or EGTA) and the calciumtransients are measured.

Methods for Treatment of Cardiac Diseases or Disorders

In other embodiments, methods are provided for treating a subjectsuffering from a disease or a disorder, such as myocardial injury, oralleviating the symptoms of such a disorder, by administering cellsisolated and cultured according to the methods disclosed.

In one embodiment, spoc cells are isolated as described herein and atherapeutically effective amount of spoc cells is administered to thesubject. In another embodiment, spoc cells are isolated anddifferentiated into cardiomyocytes, as disclosed above, and atherapeutically effective amount of the differentiated cells areadministered to a subject, such as a human. The cells may beadministered in any fashion, for example in a dose of, for example0.25-1.0×10⁶ cells. Different dosages can of course be used depending onthe clinical circumstances. The cells may be administered systemically(for example intravenously) or locally (for example directly into amyocardial defect under echocardiogram guidance, or by directapplication under visualization during surgery). In another example, thecells are administered in a gel matrix (such as Gelfoam from UpjohnCompany) which polymerizes to form a substrate in which the administeredcells can grow.

In one embodiment the subject has a myocardial injury. The myocardialinjury may be due to trauma that occurred as the result of an object orprojectile, such as a knife or a bullet, having penetrated themyocardium, or as a consequence of surgery to remove, for example, atumor. Myocardial injury may also result from diseases such ascardiomyopathy, myocardial infarction, or congenital heart disease. Inanother embodiment the subject is suffering from cardiac dysfunctionwhich includes, for example, abnormal or improper functioning of theheart valves, or abnormal communication between the chambers of theheart.

In one embodiment the spoc cells or differentiated cardiomyocytes areadministered systemically by injection. Specific, non-limiting examplesinclude administration by subcutaneous injection, intramuscularinjection, or intravenous injection. If administration is intravenous,an injectible liquid suspension of spoc cells can be prepared andadministered by a continuous drip or as a bolus.

In another embodiment, the spoc cells or differentiated cardiomyocytesare administered locally. One specific, non-limiting example of localadministration is intra-cardiac muscle injection. For intra-cardiacinjection, the spoc cells are in an injectible liquid suspensionpreparation or in a biocompatible medium which is injectible in liquidform and becomes semi-solid at the site of damaged myocardium. Aconventional intra-cardiac syringe or a controllable endoscopic deliverydevice can be used so long as the needle lumen or bore is of sufficientdiameter (e.g. 30 gauge or larger) that shear forces will not damage thespoc cells.

In other embodiments the cells are administered locally on a supportmedium. One specific, non-limiting example of a support medium is asterile mesh, or matrix, upon which the cardiomyocytes are cultured. Alayer of cardiomyocytes, for example a confluent layer ofcardiomyocytes, cultured on such a matrix can be applied locally, orgrafted at or near, a site of myocardial injury. In one embodiment thesupport medium is a biodegradable mesh. In another embodiment thesupport medium is not biodegradable. The size of the mesh, and thedensity of cells on it, can vary depending on the myocardial defectbeing treated.

In another embodiment the cells are encapsulated prior toadministration, such as by co-incubation with a biocompatible matrixknown in the art. A variety of encapsulation technologies have beendeveloped (e.g. Lacy et al., Science 254:1782-84, 1991; Sullivan et al.,Science 252:7180712, 1991; WO 91/10470; WO 91/10425; U.S. Pat. No.5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538). Duringopen surgical procedures, involving direct physical access to the heart,all of the described forms of spoc cell delivery preparations areavailable options.

The cells can be repeatedly administered at intervals until a desiredtherapeutic effect is achieved.

Use of Spoc Cells Produced to Screen Agents that Affect CardiomyocyteDifferentiation or Function

In other embodiments, methods are provided for screening agents thataffect cardiomyocyte differentiation or function. According to thismethod, a population of cardiomyocytes or their precursors is producedas described above. The population of cells is contacted with an agentof interest, and the effect of the agent on the cell population is thenassayed. The effect on differentiation, survival, proliferation, orfunction of the cells is assessed.

The methods described herein can be used to assess the effect of anagent on cardiomyocyte differentiation. In order to assess the effect ofa test agent on cardiomyocyte differentiation or function, the agent iscontacted either to spoc cells or CS cells. In several embodiments thespoc cells are maintained in medium including a growth factor betweenabout 1 day to about 8 days, between about 4 days to about 7 days, orabout 7 days before the addition of an agent.

In another embodiment the growth factor is removed from the medium,generating CS cells, at or before the agent is added. In severalspecific, non-limiting examples CS cells are maintained in the mediumbetween about 1 day to about 56 days, between about 7 days to about 28days, or between about 14 days to about 21 days before the addition ofan agent.

Differentiation of spoc cells contacted with an agent can be assessed byany means known to one of skill in the art. In one embodiment themorphology is examined, for example electron microscopy is used toassess the ultrastructure of the cells. Suitable parameters forevaluation include, but are not limited to the evaluation of gapjunctions between contacting cardiomyocytes. In other embodiments,immunohistochemical or immunofluorescence techniques are used to assessdifferentiation. In yet another embodiment, differentiation is assessedby analysis expression of specific mRNA molecules expressed incardiomyocytes. Suitable assay systems include, but are not limited toRT-PCR, in situ hybridization, Northern analysis, or RNase protectionassays. In a further embodiment the levels of polypeptides expressed indifferentiated cardiomyocytes are assayed. Specific, non-limitingexamples of polypeptide assays of use include Western blot analysis,ELISA assay, or immunofluorescence. Alternatively, calcium transientsare measured, as described above.

The assay can also be used to screen the effect of an agent oncardiomyocyte function. Any method known to one of skill in the art canbe utilized to assess cardiac function. In one embodiment the beatingrate of a cardiomyocyte is assayed to identify agents that increase ordecrease beating. One method for assessing the beating rate is toobserve beating under a microscope. Agents that can be screened in thismanner include inotropic drugs, such as sympathomimetic agents.

In one embodiment, cells contacted with the agent are compared with acontrol. Suitable controls include spoc or CS cells not contacted withthe agent, or contacted with vehicle alone. Standard values can also beused as a control.

Kits

The cells described herein are ideally suited for the preparation of akit. The kit can include a carrier means, such as a box, a bag, orplastic carton. In one embodiment the carrier contains one or morecontainers such as vials, tubes, and the like that include a sample ofspoc cells. In another embodiment, the carrier includes a container withan agent that affects differentiation, a buffer, or a vehicle for theintroduction of the cells. Instructions can be provided to detail theuse of the components of the kit, such as written instructions, videopresentations, or instructions in a format that can be opened on acomputer (e.g. a diskette or CD-ROM disk). These instructions indicate,for example, how to administer the cells to treat a myocardial defect orhow to use the cells to screen test agents of interest (such asinotropic drugs).

Without further elaboration, it is believed that one skilled in the artcan, using this description, utilize the present invention to itsfullest extent. The following examples are illustrative only, and notlimiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES Example 1 Method of Isolating and Expanding CardiomyocytePrecursor Cells from Adult Mouse Skeletal Muscle

Skeletal muscle tissue from hind legs of 6-10 week-old male C57B1/SJ6mice was cut into small pieces and digested with collagenase for twohours at 37° C. The digested tissue was cleared of cell debris and otherundigested tissue fragments by passage through a 100 μm filter and thenthrough a 40 μm filter (Falcon). The cell suspension was centrifuged atlow speed (1,400 rpm) to clear as much as of the small muscle fiberfragments as possible. The cells at this stage consisted mostly ofclusters of small round cells approximately 4 μm in diameter, calledspoc (skeletal-based precursors of cardiomyocytes) cells.

The spoc cells were plated at a density of approximately 10⁵ cells percm² in regular tissue culture dishes in complete growth medium (1:1DMEM/F 12 supplemented with 5% fetal bovine serum (FBS), 10 ng/ml humanEGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 μg/ml insulin, 5 μg/mltransferrin, 6 ng/ml selenium, 2 μg/ml ethanolamine (ITS-X, InvitrogenCorporation), 25 μg/ml gentamicin and 2.5 μg/ml fungizone (LifeTechnologies)). After a few days, the culture consisted of a floatingpopulation of round cells and some adherent fibroblasts. The round cellsenlarged as they underwent a few rounds of cell division during whichtime they became clusters of floating round cells with an increaseddiameter of 10-14 μm. The cells in these clusters, were referred to asCS (cardiac precursors from spoc) cells.

Example 2 Method of Differentiating Spoc Cells into Cardiomyocytes

CS cells were gently collected after seven days of growth in completegrowth medium. The cells were then plated in the same medium in theabsence of EGF and bFGF (differentiation medium) and were maintained at37° C. To assess the progression of differentiation of the cells, thecultures were observed at various time points using an inverted lightmicroscope. Beating frequency measurements of the cardiomyocytes wereobtained by video microscopy.

Under the differentiation culture conditions the cells gradually beganto attach to the culture dish, and elongate in shape, taking on theappearance of myoblasts. Within a few days of being maintained in thedifferentiation medium, the cells began spontaneously beating. Elongateduninucleate cells (60 μm in length) and round uninucleate cells (15 μmin diameter) both exhibited spontaneous beating. By four days postreplating the beating cells were more numerous. The beating cells didnot undergo any more cell divisions and were maintained in this mediumfor several weeks, with the maintenance of the spontaneous beatingphenotype. Spontaneous beating was continuous and measured at afrequency of 1-8 Hz. Small contractions observed in a day 14 cell (30 μmin length) were likely the consequence of an immature contractileapparatus (FIG. 1C). Cells kept at room temperature beat continuouslyfor at least 3 hours.

Example 3 Detection of Cardiac-Specific Polypeptides byImmunofluorescence

The specimens were air-dried for 30 minutes and then fixed in 4%paraformaldehyde at 4° C. followed by a rinse for 5 minutes withphosphate buffered saline (PBS). They were blocked with goat serum for30 minutes and then incubated overnight, at 4° C., with either GATA-4(mAb H-112, Santa Cruz Biotechnology), sarcomeric myosin (MF-20 Ab,ATTC), cardiac-specific troponin-T (mAb RDI-TRK4T19-1A1 1, MolecularProbes, Inc.), cardiac L-type calcium channel (mAB AB5412-2000U1a,Chemicon Inc.), cardiac-specific transcription factor Nkx2.5 (mAb N-19,Santa Cruz Biotechnology), or connexin 43 (mAb 71-07000, ZymedLaboratories Inc.) (1:200). Following the overnight incubation, thespecimens were rinsed 3 times (5 minutes each) with PBS and blockedagain with goat serum for 30 minutes. The specimens were then incubatedat room temperature with a secondary antibody, conjugated with eitherFluorescein Isothiocyanate (FITC), Texas Red, or TetramethylrhodamineIsothiocyanate (TRITC), for 1 hour. They were again rinsed 3 times (5minutes each) with PBS and then visualized with a laser confocalmicroscope (Leica) to detect fluorescent signals.

The earliest time of GATA-4 expression is after 3 days in culture ingrowth factor containing medium. Within 3 days after replating the cellsin differentiation medium, some cells begin to express sarcomericmyosin. Cytospins of day 7 CS cells stained with monoclonal antibodiesdemonstrate the expression of cardiac-specific transcription factorGATA-4, sarcomeric myosin, and cardiac-specific troponin-T. Day 14 cellsstained for GATA-4 and sarcomeric myosin. Overlays of images of cellsstained with GATA-4 and sarcomeric myosin demonstrated that they wereco-localized in the cell. At this early stage in development some cellsmay either be positive for GATA-4 or sarcomeric myosin. By day 28, themajority of cells express both proteins. By day 21 the cells arepositive for cardiac L-type calcium channel, cardiac-specifictranscription factor Nkx2.5, and connexin 43.

Example 4 Ultrastructure of Differentiated Cardiomyocytes

For routine transmission electron microscopy, cells were fixed in situon Petri dishes with 1.25% glutaraldehyde in 0.1 M cacodylate buffercontaining 1% CaCl₂ at 4° C. for 2 hours. Following fixation, cells werewashed three times in Sabatini's solution (0.1 M cacodylate buffercontaining 6.8% sucrose), and post-fixed with 1% osmium tetroxide incacodylate buffer for one hour. After three washes in Sabatini'ssolution, samples were dehydrated in alcohol and embedded in Scipoxy 812(Energy Beam Sciences, Inc. Agawarm, Mass.). Polymerization was carriedout at 37° C., for 24 hours and then at 60° C., overnight. Ultra-thinsections were cut with a Leica Ultracut UCT ultramicrotome, stained withuranyl acetate and Reynold's lead citrate, and examined with a JEOL 1200CXII transmission electron microscope.

In FIG. 1, transmission electron micrographs show the progression of CScells. At day 3 round cells with disordered myosin filaments (FIG. 1A)and large central nuclei surrounded by copious mitochondria (FIG. 1D,box and detail) exist. By day 7 elongated cells (FIG. 1E) contain densebodies (FIG. 1E arrowhead and FIG. 1B, lower box). Myosin filaments ofcharacteristic 1.6 μm-length (FIG. 1B, top box) radiate outward. A day14 cell (FIGS. 1C and F) with a single, central nucleus shows astretching out of the dense bodies (FIG. 1C) into an organizingsarcomere. By day 56, a well-defined sarcomere (FIG. 1G) is present,with identifiable A- and I-bands and Z-lines.

Example 5 Calcium Transients as a Measure of CardiomyocyteDifferentiation

Cardiomyocytes were incubated for 30 minutes at 37° C., with fluo-3 orfluo-4 dye at a concentration of approximately 5-10 μm in DMEM/F-12(dyes dissolved in DMSO 1:1 with pluronic solution). The cells were thenwashed with fresh DMEM/F-12. The images were collected with a ZeissLSM-510 laser scanning confocal system and a C-Apochromat 63× objective(1.2 N.A.). Fluo-3 and fluo-4 were excited at 488 nm with an argon laserand the emission light was collected using an LP 505 filter. The pinholewas adjusted to produce a 5 μm slice to minimize the influence of axialmovements with contraction on viewing the calcium transients. Alltransmitted light images were collected simultaneously using atransmitted light detector in conjunction with the 488 nm excitationlight. Data depth for the images was 8-bit.

Calcium transients can be observed with confocal microscopy in fluo-3-and fluo-4-treated cells (FIG. 2). Fluorescent intensity is proportionalto the amount of calcium binding to fluo-3 dye upon release of calciumfrom the sarcoplasmic reticulum. FIG. 2A shows a graphicalrepresentation of the calcium transient in a beating CS cell-derivedcardiomyocyte. Peak intensity and baseline are shown in FIG. 2B and FIG.2C, respectively. In some CS cells, calcium transients can be seenbefore observable contractions are noted, suggesting the development ofcardiomyocyte excitation elements in advance of maturing contractileelements.

Example 6 Distinguishing Spoc Cells from Bone Marrow Cells

Spoc cells are c-kit-, distinguishing them from the c-kit+bone marrowcells that have been used directly or indirectly in experiments toreconstitute infarcted heart. Despite this, spoc cells could be derivedfrom circulating bone marrow cells that become c-kit⁻ after migration toskeletal muscle. In order to more fully evaluate this question, wholebone marrow was fractionated into c-kit+ and c-kit− populations. Bothseparate and combined populations were cultured under the sameconditions as spoc cells. None of the 3 marrow cell populationsdeveloped into spontaneously beating cells.

To test whether marrow cells have the potential to differentiate intocardiomyocytes in the presence of soluble factors released from spoccells, equal proportions of marrow and spoc cells were co-cultured in aCostar transwell system, in which the two chambers are separated by a0.4 μm permeable membrane. Although the total number of cells increasedin each compartment, the spoc cells alone differentiated into beatingcells expressing cardiac markers.

In order to test if cell-cell contact between bone marrow and spoc cellswould lead bone marrow cells to differentiate into cardiomyocytes, totalbone marrow was mixed in equal proportion with EGFP-expressing spoccells obtained from EGFP-expressing transgenic mice (ACTbEGFP, TheJackson Laboratory). In three separate experiments, under the sameculture conditions, total cell number increased, but onlyEGFP-expressing cells developed into beating cells. The converseexperiments showed a similar increase in cell number, but beating cellsdid not express EGFP. Taken together, these experiments show that bonemarrow does not contain any cell population phenotypically similar tospoc cells isolated from skeletal muscle.

Example 7 Distinguishing Spoc Cells from Cells Derived from the Heart

In order to determine if spoc cells can be isolated from heart, as wellas skeletal muscle, the two tissues from the same mouse were dissociatedand cultured separately. Only the spoc cell preparation from skeletalmuscle differentiated into beating cells expressing cardiac markers. Tworeplicate co-culture experiments of both cell populations in Costartranswell systems produced an increased number of cells in bothchambers, but again, only the skeletal muscle-derived cells developedinto beating cells expressing cardiac markers.

Example 8 Distinguishing Spoc Cells from Mesenchymal Stem Cells

To determine if spoc cells can be distinguished from mesenchymal stemcells (MSC), MSC were compared to spoc cells in culture. The MSC(Clonetics Corporation) were cultured in parallel with spoc cellsgenerated from skeletal muscle as described in the methods above. TheMSC adhered to the plate almost immediately upon plating, remainedadherent throughout 12 days of observation, and did not show any sign ofbeating. In contrast, the cardiac progenitor cells from skeletal musclewere smaller in size, remained nonadherent while they developed intofloating clusters of spoc cells, and they progressed to beating cardiacmyocytes expressing cardiac markers. Spoc cells did not form in the MSCcultures. Thus, spoc cells are not MSC.

Example 9 In Vivo Differentiation of Spoc Cells

In order to determine if spoc cells are capable in vivo of continuingalong the same differentiation pathway observed in vitro, approximately100,000 EGFP-expressing, GATA-4-negative spoc cells were injected viatail vein into a mouse two months after an induced myocardial infarction(created by left coronary artery ligation). Two weeks later, histologicexamination of the heart showed EGFP positive cells that were now alsoGATA-4 positive, located in the peripheral region of the infarct. Sincespoc cells are not GATA-4 positive, these findings indicate that thesecells can home to an area of cardiac damage and begin to differentiateinto cardiomyocytes, as they do in vitro.

Example 10 Distinguishing Spoc Cells from Satellite Cells

Spoc cells and satellite cells are air-dried on glass slides for 30minutes and then fixed in 4% paraformaldehyde at 4° C. followed by arinse for 5 minutes with PBS. The cells are blocked with goat serum for30 minutes and then incubated overnight, at 4° C., with rabbit anti-met(1:200, Santa Cruz Biotechnology). Following the overnight incubation,the slides are rinsed 3 times (5 minutes each) with PBS and blockedagain with goat serum for 30 minutes. The cells are then incubated atroom temperature with a secondary antibody, conjugated with FluoresceinIsothiocyanate (FITC) for 1 hour. They are again rinsed 3 times (5minutes each) with PBS and then visualized with a laser confocalmicroscope (Leica) to detect fluorescent signals. Of the two cell typesexamined, only the satellite cells are positively stained with c-metindicating that satellite cells express c-met on their cell surface,whereas spoc cells do not.

Example 11 Method of Isolating Cardiomyocyte Precursor Cells from AdultHuman Skeletal Muscle

Skeletal muscle tissue is surgically obtained from the deltoid muscle ofan adult human, is cut into small pieces and is digested withcollagenase for two hours at 37° C. The digested tissue is cleared ofcell debris and other undigested tissue fragments by passage through a100 μm filter and then through a 40 μm filter. The cell suspension iscentrifuged at low speed to clear as much as of the small muscle fiberfragments as possible. The cells at this stage consist mostly ofclusters of small round cells approximately 4 μm in diameter which arethe human spoc cells. These cells do not express the satellite cellsurface marker c-met.

The spoc cells are plated at a density of approximately 10⁵ cells percm² in regular tissue culture dishes in complete growth medium (1:1DMEM/F12 supplemented with 5% fetal bovine serum (FBS), 10 ng/ml humanEGF, 10 ng/ml human bFGF (PeproTech, Inc.), 5 μg/ml insulin, 5 μg/mltransferrin, 6 ng/ml selenium, 2 μg/ml ethanolamine (ITS-X, InvitrogenCorporation), 25 μg/ml gentamicin and 2.5 μg/ml fungizone (LifeTechnologies)). After a few days, the culture consists of a floatingpopulation of round cells and some adherent fibroblasts. The round cellsenlarge as they undergo a few rounds of cell division during which timethey become clusters of floating round cells with an increased diameterof 10-14 μm. The cells in these clusters were referred to as CS (cardiacprecursors from spoc) cells.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiments are only examples of the invention and should not be takenas a limitation on the scope of the invention. Rather, the scope of theinvention is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

1. A method of isolating a c-kit⁻/c-met⁻ cardiomyocyte precursor cell ofmuscular origin, comprising: separating cells of less than 40 μm indiameter from a suspension of muscle cells; culturing the cells in atissue culture medium on a solid substrate; and isolating the cells insuspension in the medium; thereby isolating the c-kit⁻/c-met⁻cardiomyocyte precursor cell of muscular origin.
 2. The method of claim1, wherein separating cells of less than 40 μm in diameter from asuspension of cells comprises: passing the suspension of cells through afirst filter with a pore size of about 50-200 μm to collect a firsteluate containing cells of greater than about 50 μm and less than about200 μm in diameter; and passing the first eluate through a second filterwith a pore size of about 40 μm to collect a second eluate containingcells of less than about 40 μm in diameter.
 3. The method of claim 2wherein the first filter has a pore size of at least 100 μm and thesecond filter has a pore size of about 40 μm.
 4. The method of claim 1,wherein the tissue culture medium is a growth medium.
 5. The method ofclaim 4, wherein the growth medium is supplemented with a growth factor.6. The method of claim 5, wherein the growth factor is EGF, or bFGF, ora combination thereof.
 7. The method of claim 6, wherein the growthfactor EGF is present at a concentration between about 5 and 50 ng/ml.8. The method of claim 7, wherein the growth factor EGF is present at aconcentration between about 5 and 10 ng/ml.
 9. The method of claim 7,wherein the growth factor EGF is present at a concentration of about 10ng/ml.
 10. The method of claim 6, wherein the growth factor bFGF ispresent at a concentration between about 5 and 50 ng/ml.
 11. The methodof claim 10, wherein the growth factor bFGF is present at aconcentration between about 5 and 10 ng/ml.
 12. The method of claim 10,wherein the growth factor bFGF is present at a concentration of about 10ng/ml.
 13. A method for differentiating a c-kit⁻/c-met⁻ cardiomyocyteprecursor cell of muscular origin, comprising: separating cells of lessthan 40 μm in diameter from a suspension of muscle cells; culturing thecells in a tissue culture medium in the presence of a growth factor on asolid substrate; isolating the cells in suspension in the medium; andremoving the growth factor, thereby differentiating the c-kit⁻/c-met⁻cardiomyocyte precursor cell of muscular origin into a cardiomyocyte.14. The method of claim 13, wherein the cardiomyocyte is spontaneouslybeating.
 15. The method of claim 13, wherein the growth factor is EGF,or bFGF, or a combination thereof.
 16. The method of claim 15, whereinthe growth factor EGF is present at a concentration between about 5 and50 ng/ml.
 17. The method of claim 16, wherein the growth factor EGF ispresent at a concentration between about 5 and 10 ng/ml.
 18. The methodof claim 16, wherein the growth factor EGF is present at a concentrationof about 10 ng/ml.
 19. The method of claim 15, wherein the growth factorbFGF is present at a concentration between about 5 and 50 ng/ml.
 20. Themethod of claim 19, wherein the growth factor bFGF is present at aconcentration between about 5 and 10 ng/ml.
 21. The method of claim 19,wherein the growth factor bFGF is present at a concentration of about 10ng/ml.
 22. A mammalian cardiomyocyte differentiated from a c-kit⁻/c-met⁻cardiomyocyte precursor cell of muscular origin according to the methodof claim
 13. 23. A method of treating a myocardial injury in a subject,comprising administering a therapeutically effective amount of anisolated mammalian c-kit⁻/c-met⁻ cardiomyocyte precursor cell ofmuscular origin, thereby treating the myocardial injury.
 24. The methodof claim 23, wherein the cells are introduced locally into themyocardial injury.
 25. The method of claim 23, wherein the cells areintroduced systemically into the subject.
 26. The method of claim 25,wherein the cells are introduced intravenously.
 27. The method of claim23, wherein the myocardial injury is cardiomyopathy, myocardialinfarction or congenital heart disease.
 28. A method of treating cardiacmuscle dysfunction, comprising administering to a subject with cardiacdysfunction a therapeutically effective amount of mammalianc-kit⁻/c-met⁻ cardiomyocyte precursor cells of muscular origin thatdifferentiate into beating cardiomyocytes.
 29. The method of claim 28,wherein the cardiac muscle dysfunction is a myocardial infarction, acardiomyopathy, or a congenital heart disease.
 30. A method forscreening for an agent to determine the effect of the agent on acardiomyocyte comprising: providing mammalian c-kit⁻/c-met⁻cardiomyocyte precursor cells of muscular origin; contacting the cellswith the agent; and observing the effect of the agent on the cells. 31.The method of claim 30, wherein observing the effect comprisesdetermining the effect of the agent on differentiation of the cells. 32.The method of claim 31, wherein determination of the effect ondifferentiation comprises assaying expression of GATA-4, expression ofcardiac troponin-T, expression of L-type calcium channel, or expressionof Nkx2.5, or a combination thereof.
 33. The method of claim 31, whereinobserving the effect comprises assaying a parameter of cardiomyocytefunction of the cells.
 34. The method of claim 33, wherein the parametercomprises spontaneous beating of the cells.